Nisin, a member of the group of bacteriocins known as lantibiotics, is an antimicrobial polypeptide produced by certain strains of Lactococcus lactis (formerly Streptococcus lactis). It is manufactured through the pure-culture fermentation of these bacteria with subsequent purification and drying. The first reported use of nisin as a food preservative occurred in 1951 when it was used to control the growth of Clostridium butyricum and C. tyrobutyricum in cheese (Hirsch et al, 1951, J. Dairy Research 18:205-206).
The term "lantibiotics" was coined by Schnell et al. (1988. Nature 333:276-278) to describe a group of bacteriocins including nisin which contain the amino acid lanthionine and other "non-protein" amino acids. The common properties of these bacteriocins are reviewed by Kellner et al. (1988. Eur. J. Biochem 177:53-59) wherein they note that there ". . . polycyclic polypeptide antibiotics possess a high content of unsaturated amino acids (dehydroalanine,m dehydrobutrine) and thioether amino acids (meso-lanthionine, (2S,3S,6R)-3-methyllanthionine). Furthermore, lysinoalanine, 3-hydroxyaspartic acid and S-(2-aminovinyl)-D-cystine [are] found in some members." Members of this group include nisin, subtilin, pep 5, epidermin, gallidermin, cinnamycin, Ro09-0198, duramycin and ancovenin. These ribosomally synthesized peptide antibiotics contain from 19 to 34 amino acids and are produced by various microbes including Staphlococcus species, Bacillus species and Streptomyces species. In addition to their unique composition of non-protein amino acids, they can be distinguished from other polypeptide antibiotics on the basis of their specificity. Bacteriocins in general, and the lantibiotics in particular, are characterized by a very narrow spectrum of action. Thus, only a few species of bacteria are sensitive to a particular bacteriocin at practical concentrations. This is in contrast with other broad spectrum polypeptide antibiotics, such as polymixin B.sub.1 which are active against most bacteria and the "lytic peptides" discussed by Jaynes et al., in published international application wo 89/00194, which are active against most bacteria, yeasts and even mammalian cells.
Nisin occasionally occurs as a dimer with a molecular weight of about 7000. It contains several unusual amino acids including .beta.-methyllanthionine, dehydroalanine, and lanthionine among its total of 34 amino acids. There are five unusual thio-ether linkages in the peptide which contribute to its stability in acid solutions. Nisin is one of the most thoroughly characterized bacteriocins, and shares remarkable homology of structure and action with other lantibiotics, for example Subtilin and epidermin [Buchman et al 1988. J. Bio. Chem. 263 (31):16260-16266]. Recent reviews of nisin, its physical properties and uses include "Bacteriocins of Lactic Acid Bacteria", T. R. Klaenhammer, 1988. Biochimie 70:337-349, "Nisin", A. Hurst, 1981. Avd. Appl. Microbiol. 27:85-121, and U.S. Pat. No. 4,740,593. Nisin is the collective name describing several closely related substances which exhibit similar amino acid compositions, and some limited range of antibiotic activity. This phenomenon is discussed by E. Lipinska in "Antibiotics and Antibiosis in Agriculture" (M. Woodbine, Ed.) Pp. 103-130.
The use of nisin to combat L. monocytogenes has been reported by M. Doyle; "Effect of Environmental and Processing Conditions on Listeria Monocytogenes", Food Technology, 1988.42(4):169-171. This reference describes the initial inhibition of the organism's growth (for about 12 hours) and reports that L. monocytogenes may grow at a pH level as low as 5.0 and is resistant to alkaline pH with the ability to grow at pH 9.6.
Lysozymes (Muramidase; mucopeptide N-acetylmucamoylhydrolase; 1,4-.beta.-N acetylhexosaminodase, E.C. 3.2.1.17) are mucolytic enzymes which have been isolated from various sources and are well characterized enzymes. First discovered in 1922 by W. Fleming, egg white lysozyme was among the first proteins sequenced, the first for which a three dimensional structure was suggested using x-ray crystallography and the first for which a detailed mechanism of action was proposed. Its antimicrobial activity against gram positive bacteria is well documented, for example by V. N. Procter et al in CRC Crit. Reviews in Food Science and Nutrition, 1988, 26(4):359-395. The molecular weight of egg white lysozyme is approximately 14,300 to 14,600, the isoelectric point is pH 10.5-10.7. It is composed of 129 amino acids which are interconnected by four disulfide bridges. Similar enzymes have been isolated and characterized from other sources including such diverse producers as Escherichia coli bacteriophage T4 and human tears. Despite slight differences (for example, the human lysozyme has 130 amino acids) the capacity for hydrolysis of acetylhexosamine polymers remains essentially the same. Accordingly, for purposes of this invention, the term lysozyme is intended to include those cell wall degrading enzymes which have the ability to hydrolyze acetylhexosamine and related polymers.
Lysozyme is known to kill or inhibit the growth of bacteria and fungi, and is used in Europe to control the growth of the spoilage organism Clostridium tyrobutyrucum is cheese. It has also been proposed for use in a variety of other food preservation applications and has been reported to inhibit the growth of (and in some cases kill) Listeria monocytogenes (Hughey et al, 1987, Appl. Environ. Microbiol 53:2165-2170).
Published Australian patent application AU-A-18604/88 discloses the use of bacterialyzing enzyme products with N-acetylmuramidase, e.g. lysozyme, together with non-enzymatic preservatives for preserving foodstuffs. Non-enzymatic preservatives mentioned in this publication are complexing agents such as citric acid and EDTA, amino acids, particularly proteinogenic acids, such as cysteine, alanine, tyrosine and glycine and nucleosides and nucleotides such as inosine 5'-inosine monophosphate or phosphates such as tetrasodiumpyrophosphate (diphosphate), sodium tripolyphosphate (triphosphate) and polyphosphate or reddening agents such as alkali metal nitrates.
Lysozyme has been used for many years to prepare protoplasts of gram negative and gram positive bacteria to study cell membrane structure, function and interactions with various compounds. The mechanism of action of nisin was explored using protoplasts by Henning et al. (1986. International J. of Food Microbiology 3:121-134). They prepared protoplasts of Micrococcus luteus in an osmotically stabilized medium, and compared the action of nisin on these cells to the naturally occurring, cell wall deficient bacteria ("L-forms") of Proteus mirablis. Untreated cells, the protoplasts and L-forms were shown to be susceptible to nisin, demonstrating that the cell wall was not the target for nisin activity. There was no indication that lysozyme treated cells were more sensitive to nisin.
Jaynes et al., supra, describe the synergistic combination of "lytic peptides" from insect hemolymph and lysozyme for the lysis of mammalian cells infected with eucaryotic and procaryotic parasites and mention that the combination is also effective against bacteria.
In recently published international application WO89/12399 there is described a method whereby gram negative bacteria are controlled by a lanthionine containing bacteriocin in combination with a chelating agent and/or a detergent.
Recombinant DNA, protein engineering and chemical modification technologies can be used to effect subtle modifications of peptides and proteins to alter certain aspects of the molecules and to allow expression in new hosts. For example, the expression of nisin in new hosts is described in U.S. Pat. No. 4,740,593, and the genes for T4, human and egg white lysozyme as well as nisin and other "lantiotics" have been cloned. Human lysozyme has been expressed in Bacillus subtilus and Saccharomyces cerevisiae. These homologous molecules (nisin, epidermin, subtilin, etc. and T4, human and egg white lysozyme) should be considered as synonomous with nisin and lysozyme in the context of the present invention. It should not be necessary to use the pure components to achieve the synergistic effect. For example the combination of raw egg whites and nisin fermentation broth should exhibit synergistic anti-bacterial properties.
While nisin and lysozyme have been shown to individually inhibit the growth of some bacteria, it is not clear that either lysozyme or nisin by themselves can sufficiently inhibit the growth of these microorganisms when applied in economically feasible concentrations.